Биоинженерия / ТИ_печень(органы_ЖКТ) / Bioengineering_Liver_Transplantation

Bioengineering 2019, 6, 95

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©2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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to plastic stents, self-expanding metal stents have decreased mortality as well as the number of re-interventions, however, self-expanding metal stents are more expensive [4].

More recently, drug-eluting stents have been developed as an approach to maintain stent patency [11]. Both plastic and metal stents with anti-reflux valves have been explored as a means to decrease cholangitis and increase patency [12]. The use of biodegradable biliary stents has also been explored. In many clinical scenarios, biliary stents are only needed temporarily [13,14]. A biodegradable stent that dissolves allowing its remnants to be expelled into the duodenum would eliminate the need for a secondary device removal procedure, which would increase the risk for recurrent biliary occlusion or stenosis.

Despite many advances in stent development, one major problem that remains is the progressive loss of stent patency over time. Several factors contribute to this loss of patency. It is widely accepted that biofilm formation on the stent is a common cause of stent failure. Bacteria gain access to biliary stents by migrating from either the portal venous system or through the sphincter of Oddi [15]. Microorganisms may then adhere to the stent and begin to proliferate and secrete exopolysaccharides to form biofilms that resist antibiotic treatments. Biofilm creation, in turn, is thought to contribute to biliary “sludge” formation that ultimately leads to the loss of stent patency [16].

One area that remains unexplored is the development and use of 3D printed hepatobiliary stents. 3D printing is an emerging technology in medicine, and it would allow for the rapid production of custom medical products that are relatively inexpensive [17]. The application of 3D printed stents has been explored for use in many diseases. Several examples include the use of 3D printed vascular stents for percutaneous coronary intervention, airway stents for the treatment of central airway obstruction, esophageal stents for palliative care in inoperable esophageal malignancies, and ureteral stents which facilitate urine drainage from the kidney [18–21].

Cytocompatible, hydrophilic, and drug-deliverable 3D printed materials and techniques have been recently explored and hold great promise in cell culture models, medical devices, and tissue-engineered substrates [22–25]. Such materials include 3D printed polyvinyl alcohol (PVA), a water soluble polymer. PVA filaments demonstrate favorable 3D printability using fused deposition modeling (FDM) techniques which can be post-print crosslinked (XL) to form stable 3D hydrogels [26]. The 3D printed crosslinked polyvinyl alcohol (XL-PVA) method is capable of binding biomolecules, drugs, and cells of interest due to the molecular complexes formed during crosslinking. Such materials have applications in a variety of medical devices and wound care substrates. In this study, the XL-PVA 3D printing method was coupled with collagen injection molding to create hepatobiliary stents which have been engineered to support engraftment with human placental stem cells and cholangiocytes.

2. Materials and Methods

2.1. PVA Stent Fabrication and Crosslinking

Porous tubular stents (25 mm length, 5 mm external diameter, and 3 mm internal diameter) were created with free computer aided design (CAD) software (Tinker-CAD, AutoDesk, San Francisco, CA, USA). 96 square pores (1 mm × 1 mm) were created in the stent design with 1 mm spacing between each pore. PVA filaments (AquaSolve™, Formfutura, Nijmegen, Netherlands, 1.75 mm diameter) were 3D printed into the designed stent pattern at 201 ◦C using a consumer grade 3D printed (MakerBot Replicator desktop 3D printer, MakerBot Industries LLC, Brooklyn, NY, USA) with supports turned off and raft turned on. PVA 3D printed stents were immersed in distilled water briefly to fuse layers, and cross-linked (XL) by placing the stents in a gas vapor desiccator with two separate containers containing 20 mL of 6.25% glutaraldehyde (GA) (EMD Millipore Corporation, Darmstadt, Germany) and 10 mL of concentrated hydrochloric acid (HCl) (Fisher Scientific, Hampton, NH, USA) at 42 ◦C for 24 h. XL-PVA stents were next rinsed extensively in distilled water and soaked in 70% ethanol for 24 h. XL-PVA stents were rinsed again and placed in 1X phosphate buffer solution (PBS) for storage.

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2.2. Collagen Injection Mold Chamber Fabrication

Cylindrical injection mold chambers and stent lumen maturation plugs were designed with free CAD software (Tinker-CAD) and 3D printed on a consumer grade stereolithography 3D printer (Formlabs, Form2, Somerville, MA, USA) using flexible resin (Formlabs) with supports turned on. 3D printed parts were rinsed in 70% isopropyl alcohol (Form Wash, Formlabs) and then cured at 60 ◦C with shortwave ultraviolet light (UV) for 1 h (Form Cure, Formlabs). Injection mold chambers and maturation plugs were next rinsed in 70% ethanol followed by a 1× PBS wash in a sterile tissue culture hood and stored there until used in experiments.

2.3. Barium Sulfate Coating and X-Ray Imaging

Polycaprolactone (PCL) (Sigma Aldrich, St. Louis, MO, USA) (molecular weight 80,000) and E-Z-HD™ barium sulfate (BS) (E-Z-EM Canada Inc., Quebec, Canada) were mixed in chloroform (CF) (Sigma Aldrich) to form 10% PCL:10% BS:80% CF mixture. Hydrated XL-PVA stents were dipped in the PCL-BS-CF mixtures on each tip’s end and the chloroform was allowed to evaporate before placing the stents back into 1× PBS. Final dry PCL-BS coatings on stent tips contained 50% PCL:50% BS. XL-PVA stents and versions with PCL-BS coatings were imaged with an OEC 9900 Elite C-Arm System X-ray (General Electric, Fairfield, CT, USA) in 10 mL of 1× PBS.

2.4. XL-PVA Swelling Analysis

For swelling studies, XL-PVA pieces (n = 5) were hydrated overnight in 1X PBS and dehydrated. Each hydrated and dehydrated form was weighed to obtain average weights. To calculate mass change percentage ( M%), the average hydrated weight (M1) was subtracted from the average dry weight (M2), then divided by the hydrated weight (M1) and multiplied by 100. The mass swelling ratio was calculated by taking the ratio between the average mass of the hydrated XL-PVA (M1) and the average mass of the dehydrated XL-PVA (M2), where M1 is divided by M2. The results were statistically analyzed by using Student’s t-test and the data were expressed as the mean ± standard deviation (SD). A p-value of <0.05 was considered statistically significant.

2.5. Collagen Preparation

Rat tail type 1 collagen matrices were prepared by a modification of the protocol previously published by Benoit et al. [27]. Briefly, rat tail tendons were manually excised, washed with 100% isopropanol (Thermo Fisher Scientific, Waltham, MA, USA) and dissolved in sterile 4 mM acetic acid (Sigma Aldrich) for 24 h at 4 ◦C under constant agitation. Collagen solution was filtered through a 250 μm nylon filter (Spectrum Labs, Rancho Dominguez, CA, USA), centrifuged at 19× g for 20 min at 4 ◦C and snap frozen. Using a bench-top manifold freeze-dryer (Millrock Technology, Kingston, NY, USA), frozen aliquots were dried and stored at −20 ◦C for future use. Twenty-four hours prior to experiments, freeze-dried collagen was resolubilized in cold 0.012 M hydrochloric acid (HCl) (Sigma Aldrich) at 2.5 mg/mL final collagen concentration and incubated overnight at 4 ◦C with gentle agitation. On the day of the experiment, 0.8 mL of cold 5X PBS was added to 3.2 mL of dissolved collagen gel and the pH was titrated with 0.5 M sodium hydroxide (NaOH) (Sigma Aldrich) to 7.4.

2.6. Isolation of Mesenchymal Stem Cells

Human placentas were collected from normal term pregnancies. Collection of human placenta for mesenchymal stem cell isolation was approved by the Institutional Review Board (IRB) (approval number CR00001345_STUDY00000614). Human placental mesenchymal stem cells (PMSCs) were obtained by cultivation of microvilli after elimination of villous trophoblasts. The villous tissue was dissected from different cotyledons, excluding chorionic and basal plates. After rinsing with ice-cold PBS, the villous tissue was next digested with trypsin (0.125% trypsin solution (Sigma Aldrich) containing 0.1 mg/mL DNase I and 5 (Sigma Aldrich) mM MgCl2 (Sigma Aldrich) in Dulbecco’s

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